1. Field of the Invention
The present invention relates no the detection of organisms in liquids, and, more particularly, to the spectrophotometric detection of microorganisms in aqueous solutions.
2. Description of Related Art
Water quality is an extremely important environmental issue, particularly as regards the quality of drinking water. The number and size of the particulate matter suspended in drinking water is continuously monitored after the filtration units in water treatment facilities, specifically to detect the presence of microorganisms such as enteroviruses, protozoa, and bacteria capable of presenting serious health hazards.
The enteric protozoa Cryptosporidium and Giardia are known to cause waterborne diseases, even when present in fairly dilute concentrations. Giardia is the most frequently identifiable agent responsible for such diseases in the United States; Cryptosporidium has caused outbreaks in the United Kingdom as well as the United States and is now recognized as being one of the most disinfectant-resistant waterborne pathogens known. These occurrences have emphasized the need for rapid detection techniques or contaminants in source and treated water.
Currently there is no on-line instrumentation capable of detecting, counting, and classifying specific microorganisms. The technology known in the art requires that the particulate matter suspended in the water be concentrated and then detected with the use of microscopic techniques. Such laboratory techniques include immunofluorescent assay (IFA), polymerase chain reaction (PCR), flow cytometry (FC), and cell sorting. IFA technology may be used in conjunction with microscopy for identification following labeling with specific antibodies. The limitations associated with IFA include long analysis times, an inability to detect viability and to distinguish between species, and low sensitivity. The disadvantages of PCR include environmental interference, long analysis times, and an inability to quantify organisms. FC has the disadvantages of high instrumentation costs, high level of training of personnel required, an inability to distinguish between species, and small sample volumes.
In industrial settings, detection methods include turbidity and particle counting. Turbidity is known as a technique for evaluating filter efficiency and water quality and can be used on line. Standard turbidity measurements respond to both particle size and number; therefore, they do not distinguish between the two. Liquid-borne particle counters (LPC) illuminate a very small sample volume for analysis and have not traditionally been used for on-line applications. Although LPC cannot differentiate between species, if coupled with adequate sampling strategies, particle counters can be used effectively for on-line applications.
As is known from spectroscopy theory, a measure of the absorption of a solution is the extinction coefficient, which also provides a measure of the turbidity. Spectra in the visible region of the electromagnetic spectrum reflect the presence of metal ions and large conjugated aromatic structures double-bond systems. In the near-uv region small conjugated ring systems affect absorption properties. However, suspensions of very large particles are powerful scatterers of radiation, and in the case of microorganisms, both light scattering and absorption effects are sufficiently strong to permit quantitative detection and classification. It is therefore known to use uv/vis spectroscopy to monitor purity, concentration, and reaction rates of such large molecules.
Many attempts have been made to estimate the PSD and the chemical composition of suspended particles using optical spectral extinction (turbidity) measurements. However, previously used techniques require that either the form of the PSD be known a priori or that the shape of the PSD be assumed. The present inventor has applied standard regularization techniques to the solution of the turbidity equation and has demonstrated correct PSDs of a large variety of polymer lattices, protein aggregates, silicon dioxide particles, and microorganisms.